A genomic region where the copy number of a gene per cell differs among individuals in a certain population is referred to as copy number variation (CNV). Other known forms of polymorphism in genomic DNA number include, for example, single nucleotide polymorphism (SNP), variable number of tandem repeat (VNTR) and microsatellite polymorphism. These forms of polymorphism all involve regions of 1 kbp or shorter (each repeat unit is usually about several to several tens of bases for VNTR, and about two to four bases for microsatellite polymorphism), whereas copy number variation involves longer regions not less than 1 kb and is known to lead to a change in the copy number of the whole gene. As with SNP and the like, a type of copy number variation that occurs at frequencies of 1% or more in a population is also especially referred to as copy number polymorphism (CNP) (hereinafter, the abbreviation “CNV” is used to refer to copy number variation of any frequency).
Copy number variation occurs as relatively increased or decreased copy numbers compared with controls, known as multiplication and deletion, respectively. Usually, in human and other cells, there are two copies of each gene, one being of paternal origin, and the other being of maternal origin. In some individuals, however, there is only one copy of a particular gene per cell (deletion), or there are three copies or more (multiplication). It was in 2004 when the first definite report was presented that such gene multiplication/deletion was observed as polymorphism at high frequency in the genome of humans with normal traits (Nat. Genet., 36:949-51 (2004) and Science, 305: 525-8 (2004)). Later analysis revealed that copy number variation is a relatively common form of polymorphism, accounting for about 12% of the regions of the human genome (Nature, 444: 444-54 (2006)); it was suggested that copy number variation may be widely involved in human trait variations, including disease susceptibility and drug responsiveness.
Because copy number differences in a gene influence gene product levels, some copy number variations can be associated with susceptibility to certain diseases, drug responsiveness, and adverse reactions of drugs. Additionally, a gene may contain an allele that retains normal function, an allele that exhibits increased or decreased function, and an allele that has no function; not only variation at the gene level, but also copy number variation in a particular allele can influence phenotypes. For example, multiplication of a functional allele in the CYP2D6 gene has been shown to be correlated with extremely rapid codeine metabolism that causes opioid poisoning (N. Engl. J. Med., 351:2827-31 (2004)). To apply such information as a “point-of-care (POC)” genetic testing system to personalized medicine, it is essential to develop a novel method for determining the copy numbers of genes relevant to disease susceptibility and drug responsiveness, as well as the copy number of each allele (Genome Res., 16: 949-61 (2006)).
To date, over 2000 CNV regions have been identified by a variety of methods, including BAC-array CGH (comparative genomic hybridization) (see, for example, Nature, 444: 444-54 (2006) and Am. J. Hum. Genet., 79:275-90 (2006)), an oligonucleotide array called ROMA (representational oligonucleotide microarray analysis) (Science, 305: 525-8 (2004)), fosmid paired-end sequence mapping (Nat. Genet., 37:727-32 (2005)) and SNP mapping array (Nature, 444: 444-54 (2006)), and are summarized in the Database of Genomic Variants [projects.tcag.ca/variation/]. However, deleted or multiplied genomic regions have not fully been identified due to technical limitations of these platform technologies. For example, BAC-array CGH is incapable of detecting CNV not more than 50 kb in length because of the large probe size used for detection (100 kb or more). Meanwhile, the ROMA method is of low resolution because of the narrow coverage of genome. In fosmid paired-end sequencing, it is difficult to analyze multiple samples because a large amount of sequence per sample is analyzed. Furthermore, the SNP mapping array poses some problems, including low marker density in some parts of genomic regions (Genome Res., 16: 949-61 (2006) and Annu. Rev. Genomics Hum. Genet., 7:407-42 (2006)). In recent years, high-density oligonucleotide tiling array CGH has often been used to accurately define CNV breakpoints (see, for example, Am. J. Hum. Genet., 79:275-90 (2006)). Although this method has an advantage of high resolution, the probe specificity is inadequate, particularly in homologous regions known as CNV hotspots (Am. J. Hum. Genet., 79:275-90 (2006) and Annu. Rev. Genomics Hum. Genet., 7:407-42 (2006)).
Invader assay coupled with multiplex PCR is one of the most accurate methods for SNP genotyping (Nat Biotechnol; 17: 292-296 (1999) and J. Hum. Genet., 46:471-7 (2001)), and was used in International HapMap project (Nature; 437: 1299-1320 (2005)). This assay was originally developed as an endpoint assay, and comprises measuring fluorescence intensity after an invader reaction for 15 to 60 minutes. Although this protocol gives clear and accurate results of genotyping, nothing has been suggested concerning the applicability thereof to the detection of CNV.
Cytochrome P450 2D6 (CYP2D6) is one of the most extensively studied drug metabolizing enzymes and it is involved in the biotransformation of a large number of medications of wide therapeutic use, including blockers, antiarrhythmics, opioids, antidepressant and antipsychotic agents [Pharmacogenomics J 2005; 5: 6-13]. The CYP2D6 gene is extremely polymorphic, and over 60 known allelic variants have been reported, comprising single nucleotide polymorphisms (SNPs), short insertions and deletions (Indels), gene conversions and copy number variations (CNVs), including whole gene deletion, whole gene multiplication of same types of CYP2D6 gene like CYP2D6* 1xN and CYP2D6*2xN (up to thirteen copies per individual), whole gene duplication of different type of CYP2D6 gene like CYP2D6*10-*36 (www.cypalleles.ki.se/). Approximately half of them have been reported its involvement to the enzymatic activity in vivo and/or in vitro and some of them are risk factors for treatment failure or dose-dependent drug toxicity [N Engl J Med 2004; 351: 2827-2831, Clin Chem 2004; 50: 1623-1633, J Clin Oncol 2005; 23: 9312-9318, Pharmacogenomics J 2007; 7: 257-265, Mol Ther 2008; 83: 234-242].
To investigate association between dosage effect of these polymorphisms(alleles) or haplotypes and its enzymatic activity, it has been desired to develop the system to quantify copy number of each polymorphism (allele) by combining notions of CNVs and other polymorphisms [Nucleic Acids Res 2005; 33: e183, Nat Rev Genet 2007; 8: 639-646].
Though multiple methods have been reported for SNPs, Indels, gene conversions and CNVs genotyping in CYP2D6, including long PCR based Restriction Fragment Length Polymorphism (RFLP) [Clin Chem 2000; 46: 1072-1077], Amplichip P450 [Drug Metab Pharmacokinet 2002; 17: 157-160, Ther Drug Monit 2006; 28: 673-677], pyrosequencing [Eur J Clin Pharmacol 2003; 59: 521-526], SNaPshot [Methods Mol Biol 2005; 297: 243-252] and so on, these technologies have been developed for qualitative detection of the polymorphisms, not quantitative.
The method for total gene copy number using real-time quantitative PCR has already been established and widely used [Hum Mutat 2003; 22: 476-85, J Biomed Biotechnol 2005; 005: 48-53]. On the other hand, a few methods for allele ratio, including molecular inversion probe [Nucleic Acids Res 2005; 33: e183], TaqMan SNP genotyping assays [BMC Genomics 2006; 7: 143], melting curve analysis [Clin Chem 2000; 46:1574-1582] and Mass spectrometry [Clin Chem 2005; 51: 2358-2362] have been reported. However, the possibility of application to CYP2D6 genotyping has still been unclear.